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Abstract:

The subject invention pertains to methods to enhance the therapeutic
effects of cellular or drug treatment in various diseases and disorders.
More particularly, the present invention provides methods of treating
disorders by administering CTX0E03 cells to the patient, intravenously or
intraarterially. The treatment is useful for neurodegenerative diseases,
such as stroke. The CTX0E03 cells may be cryopreserved and/or passaged
before administration into the patient. Administration of the CTX0E03
cells into stroke rat models was at or within 48 hours after stroke.
Testing of the rat models through elevated body swing test to measure of
neurobehavioral status at the time of transplant and repeated
triphenyltetrazolium chloride (TTC) staining as a measure of infarct
volume showed short term survival that provided significant protection
from the stroke.

Claims:

1. A method of treating a neurodegenerative disease or neurological
injury in a patient, comprising: administering a therapeutically
effective amount of CTX0E03 cells to the patient, wherein the
administration is performed intravenously or intraarterially.

2. The method of claim 1, wherein the CTX0E03 cells are administered at
about 1.0.times.10.sup.4 cells to about 1.0.times.10.sup.9 cells.

3. The method of claim 2, wherein the CTX0E03 cells are administered at
about 1.times.10.sup.5 to about 1.times.10.sup.7 cells.

7. The method of claim 6, wherein the CTX0E03 cells are passaged before
administration into the patient.

8. The method of claim 1, wherein the neurodegenerative disease is
stroke.

9. The method of claim 8, wherein the CTX0E03 cells are administered
within the first 7 days of the stroke.

10. The method of claim 9, wherein the CTX0E03 cells are administered
within 2 days of the stroke.

11. A method of treating a neurodegenerative disease in a patient,
comprising: administering 1.times.10.sup.7 CTX0E03 cells to the patient,
wherein the administration is performed intravenously or intraarterially.

12. The method of claim 11, further comprising administering the CTX0E03
cells in a therapeutic composition further comprising Hank's balanced
salt solution and N-acetylcycsteine.

14. The method of claim 13, wherein the CTX0E03 cells are passaged before
administration into the patient.

15. The method of claim 11, wherein the neurodegenerative disease is
stroke.

16. The method of claim 15, wherein the CTX0E03 cells are administered
within 7 days of the stroke.

17. The method of claim 16, wherein the CTX0E03 cells are administered
within 2 days of the stroke.

18. A method for repairing neural damage caused by a disease or disorder
comprising administering 1.times.10.sup.7 CTX0E03 cells to the patient,
wherein the administration is performed intravenously or intraarterially.

[0002] This invention relates to the treatment of various neural diseases
and disorders using stem cells. Specifically, the invention provides
administering the conditionally immortalized fetal neural stem cell line
CTX0E03 to treat stroke.

BACKGROUND OF THE INVENTION

[0003] Cerebrovascular disease, considered one of the top five
non-communicable diseases, affects approximately 50 million people
worldwide, resulting in approximately 5.5 million deaths per year. Of
those 50 million, stroke accounts for roughly 40 million people. Stroke
is the third leading cause of death in developed countries and accounts
for the major cause of adult disability.

[0004] Despite the significant research into stroke, there are
depressingly few effective treatments for acute stroke, with organized
stroke care, early aspirin and thrombolytic treatment being the only
proven therapeutic strategies (Dawson & Walters (2006). New and emerging
treatments for stroke. Br Med Bull. 77-78). Infarct volume increases in
the first few hours after onset of ischaemic stroke, with the infarct
gradually subsuming the ischaemic penumbra, the region where blood supply
is significantly reduced but energy metabolism is maintained because of
collateral flow. Survival of neurons in the penumbra depends on the
severity and duration of ischaemia, however prior to reperfusion a
physiological cascade occurs, which increases intracellular calcium
(Dawson & Walters (2006). New and emerging treatments for stroke. Br Med
Bull. 77-78). This cascade self-perpetuates causing acidosis, activation
of lipase, protease and free radical generation (Dawson & Walters (2006).
New and emerging treatments for stroke. Br Med Bull. 77-78).

[0005] For ischaemic and haemorrhagic stroke there are therapeutic targets
which exist only in the early hours after stroke, requiring rapid
assessment and treatment. However, studies found that only 30% of those
suspected stroke patients received a CT or other scan on the same day.

[0008] A thrombolytic agent induces or moderates thrombolysis, and the
most commonly used agent is tissue plasminogen activator (t-PA).
Recombinant t-PA (rt-PA) helps reestablish cerebral circulation by
dissolving (lysing) the clots that obstruct blood flow. It is an
effective treatment, with an extremely short therapeutic window; it must
be administered within 3 hours from onset. It also requires a CT scan
prior to administration of the treatment, further reducing the amount of
time available. Genetech Pharmaceuticals manufactures ACTIVASE® and
is currently the only source of rt-PA. Recent studies have found that the
odds of favourable outcome were 2.8 (95% CI=1.8-9.5) if tPA is
administered within 90 min and 1.6 (95% CI=1.1-2.2) between 91 and 180
min, showing that the chances of being free of handicap after stroke are
increased nearly 3-fold by thrombolytic treatment, provided it is
administered within 90 min of onset (Dawson & Walters (2006). New and
emerging treatments for stroke. Br Med Bull. 77-78).

[0009] Neuroprotective agents are drugs that minimize the effects of the
ischemic cascade, and include, for example, Glutamate Antagonists,
Calcium Antagonists, Opiate Antagonists, GABA-A Agonists, Calpain
Inhibitors, Kinase Inhibitors, and Antioxidants. Several different
clinical trials for acute ischemic stroke are in progress. Due to their
complementary functions of clot-busting and brain-protection, future
acute treatment procedures will most likely involve the combination of
thrombolytic and neuroprotective therapies. However, like thrombolytics,
most neuroprotectives need to be administered within 6 hours after a
stroke to be effective.

[0010] Oxygenated Fluorocarbon Nutrient Emulsion (OFNE) Therapy delivers
oxygen and nutrients to the brain through the cerebral spinal fluid.
Neuroperfusion is an experimental procedure in which oxygen-rich blood is
rerouted through the brain as a way to minimize the damage of an ischemic
stroke. GPIIb/IIIa Platelet Inhibitor Therapy inhibits the ability of the
glycoprotein GPIIb/IIIa receptors on platelets to aggregate, or clump.
Rehabilitation/Physical Therapy must begin early after stroke, however,
they cannot change the brain damage. The goal of rehabilitation is to
improve function so that the stroke survivor can become as independent as
possible.

[0011] Although some of the acute treatments showed promise in clinical
trials, a study conducted in Cleveland showed that only 1.8% of patients
presenting with stroke symptoms even received the t-PA treatment (Katzan
I L, et al. (2000) Use of tissue-type plasminogen activator for acute
ischemic stroke: the Cleveland area experience. JAMA, 283:1151-1158).
t-PA is currently the most widely used of the above-mentioned acute
stroke treatments, however, the number of patients receiving any new
"effective" acute stroke treatment is estimated to be under 10%. These
statistics show a clear need for the availability of acute stroke
treatment at greater than 24 hours post stroke.

[0012] For some of these acute treatments (i.e., t-PA) the time of
administration is crucial. Recent studies have found that 42% of stroke
patients wait as long as 24 hours before arriving at the hospital, with
the average time of arrival being 13 hours after stroke. t-PA has been
shown to enhance recovery of ˜1/3 of the patients that receive the
therapy, however a recent study mandated by the FDA (Albers, et al.
(2000). Intravenous Tissue-Type Plasminogen Activator for Treatment of
Acute Stroke, The Standard Treatment with Alteplase to Reverse Stroke
Study. JAMA. 283(9)) found that about a third of the time the three-hour
treatment window was violated resulting in an ineffective treatment. With
the exception of rehabilitation, the remaining acute treatments are still
in clinical trials and are not widely available in the U.S., particularly
in rural areas, which may not have large medical centers with the needed
neurology specialists and emergency room staffing, access to any of these
new methods of stroke diagnosis and therapy may be limited for some time.

[0013] The cost of stroke in the US is over $43 billion, including both
direct and indirect costs. The direct costs account for about 60% of the
total amount and include hospital stays, physicians' fees, and
rehabilitation. These costs normally reach $15,000/patient in the first
three months; however, in approximately 10% of the cases, the costs are
in excess of $35,000. Indirect costs account for the remaining portion
and include lost productivity of the stroke victim, and lost productivity
of family member caregivers (National Institute of Neurological Disorders
and Stroke, National Institutes of Health, Bethesda, Md.).

[0014] Approximately 750,000 strokes occur in the US every year, of which
about 1/3 are fatal. Of the remaining patients, approximately 1/3 is
impaired mildly, 1/3 is impaired moderately, and 1/3 is impaired
severely. Ischemic stroke accounts for 80% of these strokes.

[0015] As the baby-boomers age, the total number of strokes is projected
to increase substantially. The risk of stroke increases with age. After
age 55, the risk of having a stroke doubles every decade, with
approximately 40% of individuals in their 80's having strokes. Also, the
risk of having a second stroke increases over time. The risk of having a
second stroke is 25-40% five years after the first. With the over-65
portion of the population expected to increase as the baby boomers reach
their golden years, the size of this market will grow substantially.
Also, the demand for an effective treatment will increase dramatically.

[0016] Given the inability to effectively mitigate the devastating effects
of stroke, it is imperative that novel therapeutic strategies are
developed to both minimize the initial neural trauma as well as repair
the damage brain once the pathological cascade of stroke has run its
course.

[0017] Transplantation of stem cells has been proposed as a means of
treating stroke. Neural stem cells are important treatment candidates for
stroke and other CNS diseases because of their ability to differentiate
in vitro and in vivo into neurons, astrocytes and oligodendrocytes. The
powerful multipotent potential of stem cells may make it possible to
effectively treat diseases or injuries with complicated disruptions in
neural circuitry, such as stroke where more than one cell population is
affected. Human umbilical cord blood (HUCB) cells administered
systemically is a very effective treatment for experimental stroke in
rats. However, HUCB cellular therapy is limited for translation as a
treatment for humans because of the potential for disease and
availability of these cells.

[0018] Because of the difficulty in effectively treating patients after
stroke, there is a need in the art for methods to enhance the treatment
of stroke.

[0019] Stem cell implants have shown some degree of success in the middle
cerebral artery occlusion (MCAO) rodent model of stroke. Previous work
has demonstrated that a single intravenous injection of human umbilical
cord blood cell (hUCBC) in aged rats can significantly reduce the number
of activated microglia, and increase neurogenesis, thus improving the
microenvironment of the aged hippocamcus and rejuvenating the aged neural
stem/progenitor cells (Bachstetter A D, et al. (2008). Peripheral
injection of human umbilical cord blood stimulates neurogenesis in the
aged rat brain. BMC Neurosci. 9:22). However, hUCBC are limited by the
number of cells attainable from cord collection, which limits the
effectiveness of such a treatment. Further, it has been observed that
cells (hES-NPC) administered into the blood stream were only capable of
migrating into the CNS at less than 1% the total cells administered
(Crokcer, et al. (2011). Intravenous administration of human ES-derived
neural precursor cells attenuates cuprizone-induced CNS demyelination.
Neuropathol Appl Neurobiol. doi: 10.1111/j.1365-2990.2011.01165.x. [Epub
ahead of print]).

[0021] However, current progenitor cell treatments rely on transplant of
cells into the brain, which is an invasive and dangerous surgery. What is
needed is a simple, safe method of administering neural progenitor cells
into a patient after stroke or other neurodegenerative disease onset.

SUMMARY OF THE INVENTION

[0022] This invention is intended to overcome, or at least alleviate, one
or more of the difficulties or deficiencies associated with the prior
art. In that regard, the present invention provides methods to enhance
the therapeutic effects of cellular or drug treatment in various diseases
and disorders. Preferably, the disorder is stroke.

[0023] In that regard, the present invention fulfills in part the need to
identify new, unique methods for treating strokes.

[0024] Accordingly, a method is provided for treating a neurodegenerative
disease in a patient or repairing neural damage caused by a disease or
disorder, by administering a therapeutically effective amount of CTX0E03
cells to the patient, where the administration is performed intravenously
(IV) or intraarterially (IA). The treatment is useful for
neurodegenerative diseases, such as stroke. The CTX0E03 cells may be
administered at about 1.0×104 cells to about
1.0×109 cells, more specifically at about 1×105 to
about 1×107 cells. In particular embodiments, the CTX0E03
cells are administered at 1×107. The cells are optionally
administered in a therapeutic composition, such as a composition
comprising Hank's balanced salt solution and N-acetylcycsteine.

[0025] The CTX0E03 cells are optionally cryopreserved before use and may
also, in some variations, also be passaged before administration into the
patient. Administration of the CTX0E03 cells may be performed at any
therapeutically effective time, however, it has been found that IV or IA
administration of the CTX0E03 cells within 2 days of stroke, and more
specifically at 48 hours after stroke, unexpectedly provides ischemic
neurons with statistically significant protection from the stroke. The
elevated body swing test (EBST) as a marker of motor asymmetry was used
as a measure of neurobehavioral status at the time of transplant and
repeated at 3 days after cell implantation along with
triphenyltetrazolium chloride (TTC) staining as a measure of infarct
volume. Short term survival was also studied as an indication of the
safety of the cell transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] For a fuller understanding of the invention, reference should be
made to the following detailed description, taken in connection with the
accompanying drawings, in which:

[0027] FIG. 1 is a graph showing significant decrease in death rate
following cell transplant. Vehicle: n=8 before treatment and n=4 2 days
after; Cells: n=4 before treatment and n=4 2 days after. *p<0.05
Chi-square test

[0028] FIG. 2 is a graph showing EBST as a measure of motor asymmetry in
vehicle and cell-treated animals. Motor asymmetry was significantly
reduced in the cell-treated animals but not the vehicle. N=4 for both
groups. *p<0.05 two-tailed t-test compared with pre-treatment group.

[0029] FIG. 3 is a graph showing the comparison of mean infarct size
between vehicle and cell treated animals. Non-significant difference
between infarct size as determined by t-test. TTC staining of two
comparable sections is shown. N=4 for both groups.

[0031] FIG. 5 is a graph showing BrdU staining of proliferating cells
shown in the SGZ and SGZ/GCL, respectively, of vehicle implanted and
CTX0E03-implanted rats 2 days after transplant. The differences between
CTX0E03 and vehicle are significant for total BrdU-positive cell counts
(P<0.001) in cell- and vehicle-implanted SGZ based on the optical
fractionator method of unbiased stereological analysis. BrdU,
bromodeoxyuridine; GCL, granular cell layer; SGZ, subgranular zone.

[0032] FIG. 6 is a graph showing DCX staining of proliferating cells shown
in the SGZ and SGZ/GCL, respectively, of vehicle implanted and
CTX0E03-implanted rats 2 days after transplant. The differences between
CTX0E03 and vehicle are significant for total BrdU-positive cell counts
(P<0.005) in cell- and vehicle-implanted SGZ based on the optical
fractionator method of unbiased stereological analysis. DCX,
doublecortin; GCL, granular cell layer; SGZ, subgranular zone.

[0034] FIG. 8(A) through (C) are images showing the existence of CTX0E03
grafts in the ventricle, but not in the SGZ. Images (A) and (C) show a
number of HuNu-positive cells (indicated by the arrows) present along the
need tract (shown by the white-dotted line) and the ventricle. Few
HuNu-positive cells colocalize with BrdU-positive cells (seen in B and
dark gray structures in C). Abbreviations: BrdU, bromodeoxyuridine, HuNu,
human nuclei antigen; SGZ, subgranular zone.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0035] The present invention may be understood more readily by reference
to the following detailed description of the preferred embodiments of the
invention and the Examples included herein. However, before the present
compounds, compositions, and methods are disclosed and described, it is
to be understood that this invention is not limited to specific nucleic
acids, specific polypeptides, specific cell types, specific host cells,
specific conditions, or specific methods, etc., as such may, of course,
vary, and the numerous modifications and variations therein will be
apparent to those skilled in the art. It is also to be understood that
the terminology used herein is for the purpose of describing specific
embodiments only and is not intended to be limiting.

[0037] The term "neurodegenerative disease" is used herein to describe a
disease which is caused by damage to the central nervous system and which
damage can be reduced and/or alleviated through transplantation of neural
cells according to the present invention to damaged areas of the brain
and/or spinal cord of the patient. Exemplary neurodegenerative diseases
which may be treated using the neural cells and methods according to the
present invention include for example, Huntington's disease, amyotrophic
lateral sclerosis (Lou Gehrig's disease), lysosomal storage disease
("white matter disease" or glial/demyelination disease, as described, for
example by Folkerth R D. (1999). Abnormalities of developing white matter
in lysosomal storage diseases. J Neuropathol Exp Neurol. 58(9):887-902.
Review), multiple sclerosis, brain injury or trauma caused by ischemia,
accidents, environmental insult, etc. In addition, the present invention
may be used to reduce and/or eliminate the effects on the central nervous
system of a stroke or a heart attack in a patient, which is otherwise
caused by lack of blood flow or ischemia to a site in the brain of said
patient or which has occurred from physical injury to the brain and/or
spinal cord. Neurodegenerative diseases also include neurodevelopmental
disorders including for example, autism and related neurological diseases
such as schizophrenia, among numerous others.

[0038] The isolation, manufacture and protocols for the CTX0E03 cell line
in generating cells in the present invention is described in detail by
Sinden, et al. (U.S. Pat. No. 7,416,888). In one application of the
cells, a clinical trial for the stereotactic intracerebral administration
of CTX0E03 drug product for the treatment of stable motor disability, 6
months to 5 years after a stroke is underway in Glasgow, Scotland
(ClinicalTrials.gov, National Institutes of Health, Identifier
#NCT01151124).

[0039] The neural stem cells of the subject invention can be administered
to patients, including veterinary (non-human animal) patients, to
alleviate the symptoms of a variety of pathological conditions for which
cell therapy is applicable. For example, the cells of the present
invention can be administered to a patient to alleviate the symptoms of
neurological disorders such as stroke (e.g., cerebral ischemia,
hypoxia-ischemia); neurodegenerative diseases, such as Huntington's
disease; traumatic brain injury; amyotrophic lateral sclerosis; multiple
sclerosis (MS) and other demyelinating diseases. In a preferred
embodiment of the present invention, the cells are administered to
alleviate the symptoms of stroke.

[0040] The term "patient" is used herein to describe an animal, preferably
a human, to whom treatment, including prophylactic treatment, with the
cells according to the present invention, is provided. For treatment of
those infections, conditions or disease states which are specific for a
specific animal such as a human patient, the term patient refers to that
specific animal. The term "donor" is used to describe an individual
(animal, including a human) who or which donates umbilical cord blood or
fetal neural stem cells for use in a patient.

[0041] The term "effective amount" is used herein to describe
concentrations or amounts of components such as differentiation agents,
fetal neural stem cells, precursor or progenitor cells, specialized
cells, such as neural and/or neuronal or glial cells, blood brain barrier
permeabilizers and/or other agents which are effective for producing an
intended result including differentiating stem and/or progenitor cells
into specialized cells, such as neural, neuronal and/or glial cells, or
treating a neurological disorder or other pathologic condition including
damage to the central nervous system of a patient, such as a stroke,
heart attack, or accident victim or for effecting a transplantation of
those cells within the patient to be treated. Compositions according to
the present invention may be used to effect a transplantation of the
fetal neural stem cells within the composition to produce a favorable
change in the brain or spinal cord, or in the disease or condition
treated, whether that change is an improvement (such as stopping or
reversing the degeneration of a disease or condition, reducing a
neurological deficit or improving a neurological response) or a complete
cure of the disease or condition treated.

[0042] The terms "stem cell" or "progenitor cell" are used interchangeably
herein to refer to umbilical cord blood-derived stem and progenitor
cells. The terms stem cell and progenitor cell are known in the art
(e.g., Stem Cells: Scientific Progress and Future Research Directions,
report prepared by the National Institutes of Health, June, 2001). The
term "neural cells" are cells having at least an indication of neuronal
or glial phenotype, such as staining for one or more neuronal or glial
markers or which will differentiate into cells exhibiting neuronal or
glial markers. Examples of neuronal markers which may be used to identify
neuronal cells according to the present invention include, for example,
neuron-specific nuclear protein, tyrosine hydroxylase, microtubule
associated protein, and calbindin, among others. The term neural cells
also includes cells which are neural precursor cells, i.e., stem and/or
progenitor cells which will differentiate into or become neural cells or
cells which will ultimately exhibit neuronal or glial markers, such term
including pluripotent stem and/or progenitor cells which ultimately
differentiate into neuronal and/or glial cells. All of the above cells
and their progeny are construed as neural cells for the purpose of the
present invention. Neural stem cells are cells with the ability to
proliferate, exhibit self-maintenance or renewal over the lifetime of the
organism and to generate clonally related neural progeny. Neural stem
cells give rise to neurons, astrocytes and oligodendrocytes during
development and can replace a number of neural cells in the adult brain.
Neural stem cells are neural cells for purposes of the present invention.
The terms "neural cells" and "neuronal cells" are generally used
interchangeably in many aspects of the present invention. Preferred
neural cells for use in certain aspects according to the present
invention include those cells which exhibit one or more of the
neural/neuronal phenotypic markers such as Musashi-1, Nestin, NeuN, class
III β-tubulin, GFAP, NF-L, NF-M, microtubule associated protein
(MAP2), S100, CNPase, glypican (especially glypican 4), neuronal
pentraxin II, neuronal PAS 1, neuronal growth associated protein 43,
neurite outgrowth extension protein, vimentin, Hu, internexin, O4, myelin
basic protein and pleiotrophin, among others.

[0043] The term "administration" or "administering" is used throughout the
specification to describe the process by which cells of the subject
invention, such as fetal neural stem cells obtained from umbilical cord
blood, or more differentiated cells obtained therefrom, are delivered to
a patient for therapeutic purposes. Cells of the subject invention be
administered a number of ways including, but not limited to, parenteral
(such term referring to intravenous and intra-arterial as well as other
appropriate parenteral routes) and intrathecal administration, among
others which term allows cells of the subject invention to migrate to the
ultimate target site where needed. Cells of the subject invention can be
administered in the form of intact CTX0E03 immortalized fetal neural stem
cells. The compositions according to the present invention may be used
without cell expansion, i.e. passaging, with a mobilization agent or
differentiation agent. Administration will often depend upon the disease
or condition treated and may preferably be via a parenteral route, for
example, intravenously. In the case of stroke, the preferred route of
administration will depend upon where the stroke is, but may be directly
into the carotid artery, or may be administered systemically. In a
preferred embodiment of the present invention, the route of
administration for treating an individual post-stroke is systemic, via
intravenous or intra-arterial administration. Optionally, the fetal
neural stem cells are administered in conjunction with an
immunosuppressive agent, such as cyclosporine A or tacrolimus.

[0044] The fetal neural stem cells of the present invention can be
administered and dosed in accordance with good medical practice, taking
into account the clinical condition of the individual patient, the site
and method of administration, scheduling of administration, patient age,
sex, body weight and other factors known to medical practitioners. The
pharmaceutically "effective amount" for purposes herein is thus
determined by such considerations as are known in the art. The amount
must be effective to achieve improvement, including but not limited to
improved survival rate or more rapid recovery, or improvement or
elimination of symptoms and other indicators as are selected as
appropriate measures by those skilled in the art.

[0045] The pharmaceutical compositions may further comprise a
pharmaceutically acceptable carrier. Pharmaceutical compositions comprise
an effective number of cells, optionally, in combination with a
pharmaceutically-acceptable carrier, additive or excipient. In certain
aspects of the present invention, cells are administered to the patient
in need of a transplant in sterile saline. In other aspects of the
present invention, the cells are administered in Hanks Balanced Salt
Solution (HBSS) or Isolyte S, pH 7.4. Other approaches may also be used,
including the use of serum free cellular media. Systemic administration
of the cells to the patient may be preferred in certain indications,
whereas direct administration at the site of or in proximity to the
diseased and/or damaged tissue may be preferred in other indications.

[0046] In some embodiments, the CTX0E03 cells can be cryopreserved in a
medium described by Hope, et al. (WO/2010/064054), in order to generate a
frozen cell product that can be stably manufactured, stored and shipped
to the treatment site, thawed and used without washing or further
significant manipulation.

[0047] Pharmaceutical compositions according to the present invention
preferably comprise an effective number within the range of about
1.0×104 cells to about 1.0×109 cells, more
preferably about 1×105 to about 1×107 cells, even
more preferably about 2×105 to about 8×106 cells
generally in solution, optionally in combination with a pharmaceutically
acceptable carrier, additive or excipient.

[0048] The term "non-tumorigenic" refers to the fact that the cells do not
give rise to a neoplasm or tumor. Stem and/or progenitor cells for use in
the present invention are preferably free from neoplasia and cancer.

[0049] Thus, fetal neural stem cells, or progenitor cells are the targets
of gene transfer either prior to differentiation or after differentiation
to a neural cell phenotype. The umbilical cord blood stem or progenitor
cells of the present invention can be genetically modified with a
heterologous nucleotide sequence and an operably linked promoter that
drives expression of the heterologous nucleotide sequence. The nucleotide
sequence can encode various proteins or peptides of interest. The gene
products produced by the genetically modified cells can be harvested in
vitro or the cells can be used as vehicles for in vivo delivery of the
gene products (i.e., gene therapy).

[0050] The following written description provides exemplary methodology
and guidance for carrying out many of the varying aspects of the present
invention.

[0051] Standard molecular biology techniques known in the art and not
specifically described are generally followed as in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,
New York (1989, 1992), and in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989). Polymerase
chain reaction (PCR) is carried out generally as in PCR Protocols: A
Guide to Methods and Applications, Academic Press, San Diego, Calif.
(1990). Reactions and manipulations involving other nucleic acid
techniques, unless stated otherwise, are performed as generally described
in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs
Harbor Laboratory Press, and methodology as set forth in U.S. Pat. Nos.
4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057 and
incorporated herein by reference. In situ PCR in combination with Flow
Cytometry can be used for detection of cells containing specific DNA and
mRNA sequences (see, for example, Testoni et al., Blood, 1996, 87:3822).

[0052] Standard methods in immunology known in the art and not
specifically described are generally followed as in Stites et al. (Eds.),
Basic And Clinical Immunology, 8th Ed., Appleton & Lange, Norwalk,
Conn. (1994); and Mishell and Shigi (Eds.), Selected Methods in Cellular
Immunology, W.H. Freeman and Co., New York (1980).

[0054] All experiments were conducted in accordance with the National
Institutes of Health guidelines, and were approved by the Institutional
Animal Care and Use Committee of the University of South Florida, College
of Medicine.

EXAMPLE 1

[0055] Adult male Sprague Dawley (SD) rats (Harlan) weighing 225-250 g,
were housed in a temperature controlled room with a 12 h light/dark cycle
and given free access to food and water. A transient intraluminal
occlusion stroke model as previously described by (Yasuhara T. et al.
(2008). Intravenous grafts recapitulate the neurorestoration afforded by
intracerebrally delivered multipotent adult progenitor cells in neonatal
hypoxic-ischemic rats. J Cereb Blood Flow Metab. 28(11):1804-10. Epub
2008 Jul. 2) was used in this study. Rats were anesthetized with 5%
isoflurane (3% maintenance) and a filament embolus was introduced into
the right MCAO and secured in place for 1 hr. For the first 8 minutes,
the left MCAO was ligated to reduce collateral reperfusion that could
prevent the infarct. Laser doppler measurement of the cerebral blood flow
was used to confirm lesioning with a drop of less than 70% being
exclusion criteria. Animals were also excluded from the study
retrospectively, if on post-mortem examination of the brain, considerable
damage or scar tissue was observed, particularly cyst formation, or if
the animal died before conclusion of the study, or showed unusual
behavior, e.g. head tilt. One hour later, under anesthesia, the filament
embolus was removed from the right MCAO and the incision sutured and the
rat allowed to recover with appropriate post-operative survival
procedures

[0056] Two days after MCAO, the animals were divided into two groups
treated i.v. with either cells or vehicle. The animals were anesthetized
with 5% isoflurane (3% maintenance) and the right jugular vein was
exposed. Animals were randomly assigned to be injected with either 0.5
mls of vehicle (Hank's Balanced Salt Solution; HBSS+0.5 mM N-acetyl
cysteine; NAC) or 1×107 CTX0E03 cells in the same vehicle,
over a 1 minute period. The incision was sutured and the rat allowed to
recover with appropriate monitoring.

[0057] Cryopreserved CTX0E03 cells were thawed and plated on
laminin-coated flasks in medium as described previously (Pollock, et al.
(2006). A conditionally immortal clonal stem cell line from human
cortical neuroepithelium for the treatment of ischemic stroke. Exp
Neurol. 199(1)) and grown at 37° C., 5% CO2 to 80% confluence
before dissociation with TrypZean/EDTA [Cambrex] and Trituration solution
(0.55 mg/ml Trypsin inhibitor [Sigma], 1% HSA, 25 U/ml Benzonase [Merck]
in DMEM:F12) to neutralize the TrypZean and digest naked DNA. Following
centrifugation and a wash in DMEM:F12 (Invitrogen), the cells were
re-suspended in vehicle at a concentration of 2×104 cells/ml.

[0058] Four animals were injected with cells, whereas eight received
vehicle. Three days after transplantation, half of the vehicle-treated
animals had died compared with none of the cell-treated which was
statistically significant as revealed by chi-squared analysis, as seen in
FIG. 1. The motor asymmetry before and 3 days after transplant was found
to be significantly reduced in the cell treated rats, seen in FIG. 2.

[0059] Three days after treatment, the rats were terminally anesthetized
and perfused with cold saline. The brain was then removed and sliced into
2 mm coronal blocks. The blocks were then stained in 2%
triphenyltetrazolium chloride (TTC) in PBS for 10 minutes in the dark.
The brain slices were then fixed in 4% paraformaldehyde. The following
day, six sections of the brain, covering the striatum, were photographed
and the area of the infarct measured (lack of TTC staining) using ImageJ
(NIH) by 2 observers blinded to the treatments. The infarct size was
normalized to the contralateral hemisphere and calculated for the whole
brain. Animal survival from treatment to perfusion between vehicle and
cells was compared by chi-squared test. Infarct size was found not
significantly different between cell and vehicle-treated rats, seen in
FIG. 3. This may have been due to the sample size.

[0060] However, motor control testing did show a significant correlation
between the % of motor asymmetry and the mean % infarct size in cell. Two
days after MCAO, the surviving rats were behaviorally tested using the
Elevated Body Swing Test (EBST) to determine motor asymmetry. The rat was
held above bedding in a high-sided box by its tail and the direction the
animal turns to is monitored 20 times. An unlesioned animal would be
expected to turn left and right equally and therefore its motor asymmetry
would be 50%. This was repeated three days after treatment by an
individual blinded to the treatment and the values compared by t-test. As
infarct size increased, the percent of asymmetry was found to increase in
a linear relationship, as seen in FIG. 4. This result was not seen in
vehicle-treated animals (data not shown).

[0061] There was therefore a significant improvement in motor behavior (as
measured by EBST) and animal survival following cell transplant 2 days
after MCAO. However, a similar significant change in infarct size was not
observed, though there was a correlation with EBST (but not in the
vehicle-treated animals), suggesting a significant difference may be
present. The decreased mortality of animals treated with the CTX0E03
cells also suggests that not only is the transplantation of these cells
safe, but that the cells also provide an improved outcome.

[0062] The i.v. implantation of CTX0E03 cells two days after experimental
ischemic stroke exerts beneficial neurological effects. The grafted cells
migrated to the injured site and either integrated with host cells or
stimulated growth factor secretion to induce regenerative processes
mediating the observed functional recovery.

EXAMPLE 2

[0063] Twelve male 22-month old Fisher (F344) rats (NIA) were anesthetized
with isoflurane and placed in a stereotaxic rig. Using a Hamilton
syringe, either vehicle (n=6) or CTX0E03 cells (n=6; 4.5×105
cells in 4.5 μl) were slowly implanted intracerebroventricularly at
coordinates relative to bregma -1 mm anteriorly, +1.6 mm medially, and
-4.5 mm dorsally to each rat. The following day, the rats were injected
twice intraperitoneally with 50 mg/kg bromodeoxyuridine
(5-bromo-2-deoxyoridine, BrdU; Sigma), 8 h apart, and were transcardially
perfused with paraformaldehyde 1 day later. The brains were then removed
and cryopreserved before being cut into 40 μm sagittal sections using
a Microm cryostat (Richard-Allan Scientific, Kalamazoo, Mich.). Six
animals from each group were implanted with either vehicle or cells.

[0064] Immunohistochemical staining for BrdU (marker of proliferation),
doublecortin (DCX; immature neurons), ionized calcium-binding adaptor
molecule I (IBA-1; microglia), glial fibrillary acidic protein (GFAP,
astrocytes), and human nuclei antigen (HuNu; transplanted human fetal
cortical cells) was performed on free floating sections as described
previously (Bachstetter A D, et al. (2008). Peripheral injection of human
umbilical cord blood stimulates neurogenesis in the aged rat brain. BMC
Neurosci. 9:22). In brief, for BrdU staining, every sixth section of a
series that surrounds the hippocampus were pretreated with 50%
formaldehyde/2% SSC for 2 h at 65° C., followed by 30 min 2 N HCl
at 37° C. and a borate buffer (pH 8.5) wash for antigen retrival.
Endogenous peroxidase quenching in 0.3% hydrogen peroxide in methanol,
followed by 1 h in blocking solution (3% normal horse serum and 0.25%
Triton X-100 in 0.1 M PBS) were performed, followed by overnight
incubation with mouse anti-rat BrdU (1:50; Roche, Indianapolis, Ind.).
This was followed by a biotinylated secondary antibody (1:200; Vector
laboratories, Burlingame, Calif.) and avidin-biotin substrate (ABU kit;
Vector Laboratories) prior to diaminobenzidine substrate visualization.
The sections were then mounted and coverslipped using Permount®
mounting medium (Fisher Chemicals, NJ). DCX can be used as a marker of
migrating neurons, since it is expressed for ˜3 weeks from the
creation of a new cell and has previously been shown to be a reliable
indicator of neurogenesis (Rao M S & Shetty A K. (2004). Efficacy of
doublecortin as a marker to analyse the absolute number and dendritic
growth of newly generated neurons in the adult dentate gyms. Eur J
Neurosci. 19:234-246; Couillard-Despres S, et al. (2005). Doublecortin
expression levels in adult brain reflect neurogenesis. Eur J Neurosci.
21:1-14). DCX immunohistochemistry was performed without antigen
retrival, using horse serum and a polyclonal goat antibody (1:150;
Santa-Cruz Biotuch, CA) and the appropriate secondary antibody.

[0065] Immunofluorescence was used to compare colocalization of BrdU and
IBA-1 or BrdU and GFAP and to demonstrate colocalization of BrdU and DCX.
The 2 N HCl at room temperature was used for antigen retrival and primary
incubation consisted of rat anti-BrdU (1:400; Accurate Chemical,
Westbury, N.Y.) and the phenotype-defining primary antibodies [rabbit
anti-GFAP (1:500; Dako, Carpinteria, Calif.), or rabbit anti-IBA1
(1:1,000; Wako, Richmond, Va.) or DCX (1:150; SantaCruz Biotech, CA)],
overnight at 4° C. Visualization was achieved using the
appropriate Alexafluor-conjugated secondary antibodies (Molecular Probes,
CA) for 2 h and the sections were then mounted and coverslipped using
Vectashield (Vector Labs). The presence of the transplanted cells was
detected using the mouse monoclonal HuNu antibody (1:50; Chemicon, CA)
that is specific for human nuclei. Visualization was achieved using an
Alexafluor-conjugated secondary antibody (Molecular Probes).
Quantification and imaging of labeled cells within the SGZ region was
performed using the optical fractionator method of unbiased stereological
cell counting (West M J, et al. (1991). Unbiased stereological estimation
of the total number of neurons in the subdivisions of the rat hippocampus
using the optical fractionator. Anat Rec. 231:482-497) using a Nikon
Eclipse 600 (for BrdU+ cell) or Olympus BX 60 (for DCX+ cell) microscope
and Stereo Investigator software (MicroBrightfield, VT). For the
proliferation study, an identical virtual grid and counting frame of
dimensions 125 μm×125 μm was used to enable us to count all
the cells that were present in a section, due to the low number of BrdU+
cells observed in the aged animals. The anatomical structures were
outlined using a 10×/0.45 objective, whereas a 60×/1.40
objective was used for cell quantification. For DCX cells, the virtual
grid and counting frame were both 150 μm×150 μm. Outlines of
the anatomical structures were done using a 10×/0.30 objective,
whereas a 40×/0.75 objective was used for cell quantification.
Defining the SGZ of the dentate gyms as a two-cell diameter band on both
sides of the granular cell layer (GCL), the number of BrdU+ cells within
the SGZ was counted. DCX+ cell counts were made in the SGZ/GCL, due to
possible cell migration. To identify cell type-specific markers
co-expressed in BrdU cells, immunofluorescent colocalization was assessed
using an Olympus IX 70 microscope with a 10×/0.30, 20×/0.40
or 40×/0.60 objective and an Olympus DP 71 camera connected to a DP
manager (Olympus, Japan). These cell counts were performed in the
SGZ/GCL.

[0066] Data represent mean±SEM and statistical testing was by unpaired
two-tailed t-test using P<0.05 as significant.

[0067] Twelve aged rats were implanted with either CTX0E03 cells or
vehicle and treated with BrdU 24 h later. Forty-eight hours from the
initial implant, the animals were perfused with paraformaldehyde, their
brains removed and cryopreserved prior to sectioning sagitally at 40
μm. The sections were labeled with a number of different antibodies to
determine cell proliferation, phenotype, and survival in the SGZ of the
dentate gyms.

[0068] The presence of proliferating cells was determined using nuclear
BrdU labeling. This was evident in the SGZ of the dentate gyms in both
vehicle (218.0±31.00) and cell-treated (694.0±130.0) animals. A
3-fold significant increase in cell number was apparent in the
cell-treated rats (t=3.894; df=9; P=0.0037; n=6), seen in FIG. 5. The
presence of neuronal precursor cells was determined using DCX labeling of
the SGZ. Labeled cells were seen in both vehicle (970±32.7) and
cell-treated (1,202.4±61.9) animals, but again the number of cells was
significantly increased in the cell-treated animals compared with the
vehicle (t=4.29; df=8; P=0.002; n=5), as seen in FIG. 6.

[0069] Confirmation that the DCX cells were also BrdU-positive was
demonstrated by colocalization staining and confocal imaging, as seen in
FIG. 7. Further identification of the potential phenotype of the
proliferation cells was determined by using IBA-1 and GFAP staining for
microglial and astrocytes, respectively, with the localization of BrdU.

[0070] Immunofluorescent IBA-1- and GFAP-positive staining cells were
abundant, whereas nuclear BrdU-positive cells were rare. Thus,
colocalization of BrdU and IBA-1 was very limited, and BrdU and GFAP
co-expression was not found within the SGZ. No significant differences
could be observed between staining in the vehicle- and cell-treated
animals (data not shown).

[0071] The presence of the transplanted cells at the injection site and in
the SGZ was determined using HuNu staining. Human nuclei staining
revealed no HuNu-positive cells within the SGZ, demonstrating that none
of the BrdU-labeled cells were transplanted cells and instead were
endogenous in origin. Some HuNu staining was apparent along the needle
tract and in the ventricle, as seen in FIGS. 8(A) though (C). However, no
HuNu-positive cells were found within the SGZ of either vehicle or
cell-implanted rats, evidencing that none of the BrdU-labeled cells
within the SGZ were transplanted cells, but instrade were endogenous in
origin.

[0072] The absence of HuNu staining within the SGZ demonstrates that at 2
days from injection, the transplanted cells have not migrated to the
region to either cause the effect or differentiate into immature neuronal
cells, but instead are exerting their influence such as directly inducing
cell proliferation or indirectly reducing inflammation to stimulate cell
proliferation from the injection site. It is likely the cells are acting
through the rapid secretion of anti-inflammatory cytokines, such as
IL-10, or neurotrophic factors, such as brain-derived neurotrophic
factor, nerve growth factor, or neurotrophin-3, which have been known to
encourage the growth and differentiation of new neurons. CTX0E03 cells
were previously shown to secrete VEGF and other factors in vitro (Eve D
J, et al. (2008). Release of VEGF by ReN001 cortical stem cells. Cell
Transplant. 17:464-465). Palmer et al. (Palmer T D, et al. (2000).
Vascular niche for adult hippocampal neurogenesis. J Comp Neurol.
425:479-494) reported that in the adult rat SGZ, neurogenesis occurs in
close proximity to blood vessels, where VEGF expression is high and
angiogenesis is ongoing. Based on this and other evidence, Palmer et al.
(Palmer T D, et al. (2000). Vascular niche for adult hippocampal
neurogenesis. J Comp Neurol. 425:479-494) argued that neurogenesis and
angiogenesis might be mechanistically linked, citing VEGF as a factor
that might provide such a linkage. In addition, it has been shown that
intracerebroventricular infusion of VEGF stimulated the proliferation of
neuronal precursors in the SGZ and SVZ both in vitro and in vivo (Jin K,
et al. (2002). Vascular endothelial growth factor (VEGF) stimulates
neurogenesis in vitro and in vivo. Proc Natl Acad Sci USA.
99:11946-11950; Sun Y, et al. (2006). Vascular endothelial growth
factor-B (VEGFB) stimulates neurogenesis: evidence from knockout mice and
growth factor administration. Dev Biol. 289:329-335). Without being bound
to any specific theory, given the above observations, the effects of
CTX0E03 cells on endogenous neural proliferation may be modulated by
VEGF. This could include the use of conditioned media in which the cells
have secreted factors such as VEGF and attenuated cells for transplant,
for example cells attenuated by freeze-thaw activity or heat
inactivation. This would show that the effect is due to the factors
secreted by the cells rather than the cells themselves.

[0073] Intracerebroventricular transplantation of CTX0E03 cells into rat
brain results in increased proliferation within at least one of the
endogenous stem cell reservoirs of the brain, the SGZ. This proliferation
is of immature neuronal cells as shown by the increased DCX staining but
the absence of significant IBA-1 and GFAP colocalization with BrdU.
Confirmation that the neuronal precursors revealed by DCX staining were
also proliferative (as shown by the BrdU colocalization) was also
obtained.

[0074] While CTX0E03 cells do seem to have an effect on endogenous
neuronal proliferation, it is not clear exactly how this occurs.
Previously work has shown that reducing neuroinflammation in rats be
blocking the conversion of pro-interleukin (IL)-1β to IL-1β
through inhibition of the converting enzyme caspase-1 rescued some rats
from age-related decreases in neurogenesis (Gemma C, et al. (2007)
Blockade of caspase-1 increases neurogenesis in the aged hippocampus. Eur
J Neurosci. 26:2795-2803) and resulted in an improvement in cognitive
function, which is often affected by stroke related brain damage (Gemma
C, et al. (2005). Improvement of memory for context by inhibition of
caspase-1 in aged rats. Eur J Neurosci. 22:1751-1756). Furthermore, with
hUCBCs, exogenous stem cells stimulate the endogenous neural progenitor
cells to increase proliferation, and reduce neuroinflammation as
evidenced by a decrease in the number of activated microglia (Bachstetter
A D, et al. (2008). Peripheral injection of human umbilical cord blood
stimulates neurogenesis in the aged rat brain. BMC Neurosci. 9:22). No
significant increase in the negligible number of colocalized BrdU- and
IBA-positive cells was observed between vehicle and cells at the site of
proliferation, suggesting that neither the cells nor the injection had
induced an immune response of new microglial cells. Further, previous
work has shown that administration of human peripheral blood mononuclear
cells as a control for the effect of human umbilical cord blood
delivering cells did not alter neuronal proliferation or hippocampal
neurogenesis (Bachstetter A D, et al. (2008). Peripheral injection of
human umbilical cord blood stimulates neurogenesis in the aged rat brain.
BMC Neurosci. 9:22). As well as the observed increased neuronal
proliferation within the dentate gyms following hUCBC transplantation
(Bachstetter A D, et al. (2008). Peripheral injection of human umbilical
cord blood stimulates neurogenesis in the aged rat brain. BMC Neurosci.
9:22), glial restricted progenitors or NSCs from rats and mice have also
been shown to promote endogenous NSCs number and survival in a more
long-term study in younger rats (12 months compared with 22 months) and a
3-fold increase in cell number in the cell-transplanted animal.
(Hattiangady B, et al. (2007). Increased dentate neurogenesis after
grafting of glial restricted progenitors or neural stem cells in the
aging hippocampus. Stem Cells. 25:2104-2117).

[0075] A clonal human NSC line, CTX0E03, has conditionally immortalized
using the fusion transgene c-mycERTAM to allow controlled expansion
when cultured in the presence of 4-hydroxy-tamoxifen. No safety or
toxicology issues identified in in vivo studies with this cell line. The
data presented herein evidences an additional use of CTX0E03 cells to
promote the endogenous restorative properties of the brain.

[0076] In the preceding specification, all documents, acts, or information
disclosed does not constitute an admission that the document, act, or
information of any combination thereof was publicly available, known to
the public, part of the general knowledge in the art, or was known to be
relevant to solve any problem at the time of priority.

[0077] The disclosures of all publications cited above are expressly
incorporated herein by reference, each in its entirety, to the same
extent as if each were incorporated by reference individually.

[0078] While there has been described and illustrated specific embodiments
of an intravenous or intraarterial treatment for a neurodegenerative
disease, it will be apparent to those skilled in the art that variations
and modifications are possible without deviating from the broad spirit
and principle of the present invention. It is intended that all matters
contained in the foregoing description or shown in the accompanying
drawings shall be interpreted as illustrative and not in a limiting
sense. It is also to be understood that the following claims are intended
to cover all of the generic and specific features of the invention herein
described, and all statements of the scope of the invention which, as a
matter of language, might be said to fall therebetween.